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The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5′-untranslated region (5′-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5′-UUUCGU-3′ hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Blood-pressure-lowering drugs are proposed to foster SARS-CoV-2 infection by pharmacological upregulation of angiotensin-converting enzyme 2 (ACE2), the binding partner of the virus spike (S) protein, located on the surface of the host cells. Conversely, it is postulated that angiotensin–renin system antagonists may prevent lung damage caused by SARS-CoV-2 infection, by reducing angiotensin II levels, which can induce permeability of lung endothelial barrier via its interaction with the AT1 receptor (AT1R). Methods: We have investigated the influence of the ACE inhibitors (lisinopril, captopril) and the AT1 antagonists (telmisartan, olmesartan) on the level of ACE2 mRNA and protein expression as well as their influence on the cytopathic effect of SARS-CoV-2 and on the cell barrier integrity in a Caco-2 cell model. Results: The drugs revealed no effect on ACE2 mRNA and protein expression. ACE inhibitors and AT1R antagonist olmesartan did not influence the infection rate of SARS-CoV-2 and were unable to prevent the SARS-CoV-2-induced cell barrier disturbance. A concentration of 25 µg/mL telmisartan significantly reduced the virus replication rate. Conclusion: ACE inhibitors and AT1R antagonist showed neither beneficial nor detrimental effects on SARS-CoV-2-infection and cell barrier integrity in vitro at pharmacologically relevant concentrations.
The duration of infectivity of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) in living patients has been demarcated. In contrast, a possible SARS-CoV-2 infectivity of corpses and subsequently its duration under post mortem circumstances remain to be elucidated. The aim of this study was to investigate the infectivity and its duration of deceased COVID-19 (coronavirus disease) patients. Four SARS-CoV-2 infected deceased patients were subjected to medicolegal autopsy. Post mortem intervals (PMI) of 1, 4, 9 and 17 days, respectively, were documented. During autopsy, swabs and organ samples were taken and examined by RT-qPCR (real-time reverse transcription-polymerase chain reaction) for the detection of SARS-CoV-2 ribonucleic acid (RNA). Determination of infectivity was performed by means of virus isolation in cell culture. In two cases, virus isolation was successful for swabs and tissue samples of the respiratory tract (PMI 4 and 17 days). The two infectious cases showed a shorter duration of COVID-19 until death than the two non-infectious cases (2 and 11 days, respectively, compared to > 19 days), which correlates with studies of living patients, in which infectivity could be narrowed to about 6 days before to 12 days after symptom onset. Most notably, infectivity was still present in one of the COVID-19 corpses after a post-mortem interval of 17 days and despite already visible signs of decomposition. To prevent SARS-CoV-2 infections in all professional groups involved in the handling and examination of COVID-19 corpses, adequate personal safety standards (reducing or avoiding aerosol formation and wearing FFP3 [filtering face piece class 3] masks) have to be enforced for routine procedures.
TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.
Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection
(2021)
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
(1) Background: The aim of our study was to identify specific risk factors for fatal outcome in critically ill COVID-19 patients. (2) Methods: Our data set consisted of 840 patients enclosed in the LEOSS registry. Using lasso regression for variable selection, a multifactorial logistic regression model was fitted to the response variable survival. Specific risk factors and their odds ratios were derived. A nomogram was developed as a graphical representation of the model. (3) Results: 14 variables were identified as independent factors contributing to the risk of death for critically ill COVID-19 patients: age (OR 1.08, CI 1.06–1.10), cardiovascular disease (OR 1.64, CI 1.06–2.55), pulmonary disease (OR 1.87, CI 1.16–3.03), baseline Statin treatment (0.54, CI 0.33–0.87), oxygen saturation (unit = 1%, OR 0.94, CI 0.92–0.96), leukocytes (unit 1000/μL, OR 1.04, CI 1.01–1.07), lymphocytes (unit 100/μL, OR 0.96, CI 0.94–0.99), platelets (unit 100,000/μL, OR 0.70, CI 0.62–0.80), procalcitonin (unit ng/mL, OR 1.11, CI 1.05–1.18), kidney failure (OR 1.68, CI 1.05–2.70), congestive heart failure (OR 2.62, CI 1.11–6.21), severe liver failure (OR 4.93, CI 1.94–12.52), and a quick SOFA score of 3 (OR 1.78, CI 1.14–2.78). The nomogram graphically displays the importance of these 14 factors for mortality. (4) Conclusions: There are risk factors that are specific to the subpopulation of critically ill COVID-19 patients.
The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. However, the factors predisposing individuals to severe disease remain poorly understood. Here, we show that levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells, are elevated in SARS-CoV-2-infected Caco-2 cells, Calu-3 cells, and air−liquid interface cultures of primary human bronchial epithelial cells. Moreover, SARS-CoV-2 infection increases SIRPalpha levels, the binding partner of CD47, on primary human monocytes. Systematic literature searches further indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions that may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, age-related and virus-induced CD47 expression is a candidate mechanism potentially contributing to severe COVID-19, as well as a therapeutic target, which may be addressed by antibodies and small molecules. Further research will be needed to investigate the potential involvement of CD47 and SIRPalpha in COVID-19 pathology. Our data should encourage other research groups to consider the potential relevance of the CD47/ SIRPalpha axis in their COVID-19 research.
Background: Testing for COVID-19 with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) may result in delayed detection of disease. Antigen detection via lateral flow testing (LFT) is faster and amenable to population-wide testing strategies. Our study assesses the diagnostic accuracy of LFT compared to RT-PCR on the same primarycare patients in Austria. Methods: Patients with mild to moderate flu-like symptoms attending a general practice network in an Austrian district (October 22 to November 30, 2020) received clinical assessment including LFT. All suspected COVID-19 cases obtained additional RT-PCR and were divided into two groups: Group 1 (true reactive): suspected cases with reactive LFT and positive RT-PCR; and Group 2 (false non-reactive): suspected cases with a non-reactive LFT but positive RT-PCR. Findings: Of the 2,562 symptomatic patients, 1,037 were suspected of COVID-19 and 826 (79.7%) patients tested RT-PCR positive. Among patients with positive RT-PCR, 788/826 tested LFT reactive (Group 1) and 38 (4.6%) non-reactive (Group 2). Overall sensitivity was 95.4% (95%CI: [94%,96.8%]), specificity 89.1% (95%CI: [86.3%, 91.9%]), positive predictive value 97.3% (95%CI:[95.9%, 98.7%]) and negative predictive value 82.5% (95%CI:[79.8%, 85.2%]). Reactive LFT and positive RT-PCR were positively correlated (r = 0.968,95CI=[0.952,0.985] and κ=0.823, 95%CI=[0.773,0.866]). Reactive LFT was negatively correlated with Ct-value (r = -0.2999,p < 0.001) and pre-test symptom duration (r = -0.1299,p = 0.0043) while Ct-value was positively correlated with pre-test symptom duration (r = 0.3733),p < 0.001). Interpretation: We show that LFT is an accurate alternative to RT-PCR testing in primary care. We note the importance of administering LFT properly, here combined with clinical assessment in symptomatic patients.